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Abstract:

Ithomiinae butterflies are protected from predation by pyrrolizidine alkaloids. These compounds are acquired as adults primarily from nectars, and, in the case of females from males via spermatophores. Nectar sources are most common outside of the forest, where Ithomiine collect in large aggregations, called leks, where mating occurs. In these leks males also use PAâ€™s as precursors for mating pheromones. Visits to floral sources of protective compounds by Ithomiinae butterflies is 90-99% male (Drummond 1976). Three hypotheses explain this phenomenon. 1) females are constrained to obtain these pyrrolizidine alkaloids (PAâ€™s) from males via spermatophores passed while mating, 2) females prefer to remain in
forested mating aggregations (leks) rather than visit open areas where most nectar sources occur and predation risk is likely greater, and 3) males must visit floral sources more often, as they use PAâ€™s as
precursors for mating pheromones and then pass them to females in mating. To test these hypotheses, floral baits known to contain PAâ€™s (Ageratum cf. reedii, Asteraceae) were placed in the center of a mating lek, at varying distances away from the lek in two directions, towards pasture or primary forest. Female Ithomiines were only found within the mating lek, possibly as a result of higher risk aversion there. Male
Ithomiinae were more abundant on the A. cf. reedii than females, perhaps due to their dual use of pyrrolizidine alkaloids for both defense and mating. Abundance was highest in the center of the lek and gradually decreased as geographical distance increased. This pattern was only offset in an outlier site
approximately 1600m away from the general area of study. Although abundant, species richness in site eight was particularly low. This may suggest that species found in the outlier site were from a different population. ( ,, )

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2 abundancia fue mayor en el centro del lek y disminuyÃ³ gradualmente conforme la distancia geogrÃ¡fica aumentaba. Este patrÃ³n solo tuvo una excepciÃ³n en un sitio aproximadam ente a 1600m del Ã¡rea general de esudio. La abundancia de la riqueza de especies en el sitio ocho fue particularmente baja, sugiriendo que las especies que se encontraron en el sitio excepcional pertenecÃ­an a una poblaciÃ³n diferente. INTRODUCTION Itho miinae, a neotropical subfamily of Nymphalidae, are notorious for their MÃ¼llerian mimicry and unpalatability, which greatly deters vertebrate and invertebrate predators (DeVries 1987; Master 1992). Ithomiine butterflies are characterized for their distinc tive wing coloration, which broadcasts their unpalatability to predators. Species differ in their wing construction from transparent, translucent, or opaque, and the colors are often arrayed in a combination of orange, black, brown, or yellow (Drummond 19 76). Other butterfly families, non Ithomiines, mimic these patterns of coloration in order to present predators with visual cues of their noxious taste (DeVries 1987). Ithomiinae obtain their unpalatability from pyrrolizidine alkaloids, commonly called mechanism but as precursors for mating pheromones by males (Brown et al. 1991). It was originally assumed that Ithomiines gained chemical defense from Solanaceae compounds in th e larval foodplants (Brown 1984) but more recent studies have proven pheromones, the Ithomii nae males release them via structures called hairpencils (DeVries 1987). These hairpencils are located on the dorsal costal margin of the hindwing and are covered by the forewing (Drummond 1976). Once male Ithomiines begin to emit pheromones, both males and females of the same species, as well as different species and genera, are attracted to the area (Haber 1978; Brown et al . 1991). These aggregations of Ithomiines have been termed a lek and have approximately equal sex ratios; females make up 25 50% of the lek (Brown et al . 1991; Drummond 1976). In these leks females protect their offspring (Brown et al . 1991). Male Ithomiines are attracted to A. reedii (Asteraceae) small purple flowered species is abundantly found along roadsides dissimilar to the shady undisturbed habitat Ithomiines prefer (Drummond 1976; Haber 1978; Zuchowski 1991). Ithomiinae visitation to these flowers is 90 99% male (D rummond 1976). Given that A. reedii are found in open, disturbed areas, and males are more commonly found on the flowers, the question was raised as to why females are not visiting these flowers the first being that females females and therefore need to visit flowers containing alkaloids more frequently in order to maintain a sufficient level for both defense and mati ng purposes. Females on the other hand only need an adequate amount for egg defense. Finally, risk aversion due to increased vulnerability to predators in open areas may deter females from leaving the safety of the forest. When in the forest butterflies have the advantage of dense vegetation which makes capture by predators, such as birds, much more difficult. In open areas there is little to no vegetation to fly between and escape larger predators that cannot avoid forest obstructions. If females are

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3 than no females will be seen visiting A. reedii . On the contrary, if females can obtain A. reedii due to risk a version factors then they will be found feeding on A. reedii if the flower is located in a safe environment such as a lek. that more males will be feeding on flowers regardless of the flower location. MATERIALS AND METHODS Study area and species This study was conducted in San Luis, Costa Rica during the first two weeks of November 2004. San Luis, located at 1300m, is found in life zone one (Hayes and Laval 1989) Premontane Moist Forest (Holdridge 1967). A lek o f Ithomiinae were found along the Camino Real trail and used in this study. In order to determine a lek, an abundance of Ithomiinae species were found; this large quantity constituted the center of the lek, and individuals gradual decreased and an eventua l near nonexistence of Ithomiinae determined the edges of the lek. Based upon distance from this mating lek, seven sites were selected. A natural stand of A. reedii was also studied approximately 550m up the cement road name La Trocha leaving the San Lui s community and connecting to Monteverde. Site one was located 900m from the center of the lek (Fig. 2). This site was in pasture with tall grass and during selective days was grazed by cows and used as a holding ground for horses. In order to determine exact distance a tape measure was used. Site two was 850m from the center of the lek and located along a road with forest on either side. A barbed wire fence divided the road from the forest edge. Site three marked one edge of the lek and was approximate ly 800m from site four. Site three was located along the Camino Real trail and could be classified as secondary forest. The center of the lek, site four, could be described as old secondary growth. Site five, the other lek edge, was located in old secon dary forest growth. This site was located 800m from the center of the lek; site four. Site six was 850m from site four and could be described as primary growth. The final site in the Camino Real was site number seven and located 900m from site four; for est growth was primary. Site eight, the natural stand of A. reedii , was located approximately 150m from where A. reedii were collected for the other seven sites (Fig. 2). This outlier site on La Trocha consisted of a small patch of A. reedii . These flo wers were not picked nor put in bundles, but species of Ithomiinae were counted the same way as those found both near and in the Camino Real trail. Flower location and arrangement A. reedii were collected from La Trocha approximately 500 to 600m from th e base of the road near site eight. A. reedii were picked and placed in bundles of approximately 20 flowers, leaving a stem length of roughly 20 cm. A rubber band was then wrapped around the base of each bundle and placed in a 50mL conical vial filled wi th water. A total of seven bundles of A. reedii were made and placed in one of the seven sites. A bundle of A. reedii was then tied around a liana, tree, sapling, or fence post using red

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4 flagging tape. The flowers were set aside from other vegetation an d were approximately 1.5m off the ground. New bundles of A. reedii were collected every three days and replaced the old bundles at each site. Identification and marking of Ithomiinae From 10 AM to 2 PM, each of the seven sites was visited a total of f our times. Site eight, La Trocha roadside, was surveyed five times. Ithomiinae feeding on A. reedii were carefully pulled off the flower and placed into a butterfly envelope. If an Ithomiinae left the flower before collection, the individual was capture d using a butterfly net and then placed in a butterfly envelope. Each individual was then removed from the envelope and the species and sex were determined (DeVries 1987). Butterflies were marked on the abdomen with a site specific color and pattern usin g xylene based Ideal 563 Speedry Paint Markers before released. Marking prevented recapture and allowed tracking of individuals between sites. RESULTS Sex Ratios Three hundred and ninety six Ithomiine individuals were caught from the eight sites. Of these 15 were females. Females were only found in sites three through five; sites within the lek (Fig. 1a). No female Ithomiines were found in site eight, an outlier site. Site four had approximately ten times more males than females. Similar to site four, the ratio of males to females in site five was ten to one. Fourteen times more male Ithomiines were found than female in site three (Fig. 1a & Table 2). Abundance Individuals were most abundant in sites four, center of the lek, and eight, outli er site (Fig. 1a). As geographical distance in either direction increased away from site four, Ithomiines decreased in abundance. Comparisons of number of individuals between sites the same distance away from site four in either direction showed noteworth y trends. More individuals were found in site five than in site three, both edge sites (Fig. 1a). Site six had more visitors than site two, both located 850m from site four. Sites one and seven where both located 900m from site four (lek center), but si te seven had nearly ten times more individuals than site one. In general sites in forested area, sites three through seven, had higher abundance than those sites found in pasture or road, sites one and two, with the exception of La Trocha, site eight.

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5 Richness Thirteen Ithomiinae species were found in this study (Table 2). Site two had the highest richness, nine species, nearly five times that of site one, with just two species (Fig. 1b & Table 2). Sites three, five, and six had comparable richnes s; eight species were found in each site. Site four, seven, and eight also had similar richness, six species in site four, seven species in site seven and five in site eight. All sites were approximately two to three times more species rich than site one . Site one had comparable richness to that of the females. Females, found in site three, four, and five, had the same species richness times (Fig. 1b). Diversity Shannon Weiner diversity index was used in order to test species diversity. Overall, sites two through eight had an index between 1.94 and 1.28 (Fig. 1c). Site one had a diversity index approximately four times lower than the other seven sites. Females were only observed feeding on A. reedii in sites three though five. Females had an average diversity index of 0.66; a value 2.5 times lower than the average index of males found in sites two through eight (average =1.63). Overlap Species similarity bet ween sites was tested using a Sorenson indices overlap matrix. No particular trends were observed. Site one shared the most species with both sites two and three (Table 1). Site two had the most species similarity with site six. Species overlap was gre atest for site three when compared to site five. Sites four and six shared the most species with each other. Site five shared the most species with site six. Site seven had the most species in common with site four. Finally, site eight had the greatest overlap index when compared to sites four and five. Generally sites four and six shared the most species with other sites. Additional Observations A. reedii was approx imately 300m away from site one but upon passing the natural floral stand, Ithomiines were never observed feeding. Although individuals were marked at each site no recaptures of the same individual took place over the course of this study. DISCUSSION Two hypotheses were raised in order to explain why visits to floral sources are 90 99% male dominated. First, females are unable to use flowers as PA sources and aware and therefore prefer to stay in forested areas where floral PA sources do not occur. However, it was also hypothesized that males must visit PA sources more frequently due

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6 A . reedii . This refutes the spermataphores. In fact, feeding experiments done by Masters (1990) showed that hand ere offered sources in the form of solutions. This further shows that females are not environment in which they were raised or perhaps feel little risk is involved. When bu ndles of A. reedii were placed within the lek females were found to be feeding on the flowers. The lek was located within forested areas and Ithomiine from A. reedii , n ot indirectly via males. The majority of females were found in the center of the lek as opposed to peripheral locations 800m on either side. Although in this study females were only found within lek boundaries other studies show that females do in fact le ave the aggregation. One such reason to leave is ovipositation, which often takes place on host plants located outside of forested area (Haber 1978). During this event female Ithomiines have been observed to leave during 10am to 1pm when predators, such as birds, are least active (Haber 1978). This again suggests that females only leave the aggregations or forested area when absolutely necessary, their obligation and desire to produce offspring may constitute as one such reason. Although females do leav e the lek, this study, along with others suggests they prefer to remain in low risk areas. Therefore, if presented with PA sources in protected areas females do feed on the source. The difference in male abundance to female abundance may be explained by the pheromone purposes and pass them in spermatophores they may exhaust these compounds more quickly than females (Brown et al . 1991). For this reason, it is possible that male s need to visit flowers containing PA compounds much more frequently in order to replenish their supply. Although not supported by other data, perhaps females are attracted to males containing more compounds. For this reason males may want more nhance mating success therefore explaining the high frequency of male visitation to flower sources. It is also important to recognize that previous studies have shown that leks are 20 50% females (Drummond 1976). This, therefore, eliminates the possibil ity that males have a higher abundance due to a skewed sex ratio. Aside from explanations pertaining to differences in abundance, species richness also revealed information regarding visitation patterns. In general, the highest abundance of Ithomiinae was found in the center of the lek and slowly decreased as distance from the lek increased. In a comparison of site one and seven, the two sites 100m from the edge of the lek, one in pasture and one in primary forest respectively, ten times more individuals were found in the primary forest. It is known that Ithomiinae butterflies prefer to occupy forested area, which would help to explain this trend. However site eight, though open and far from the lek, had nearly as t is possible that the individuals at this site were from a different population and should therefore not be compared to the other seven sites. Species richness and diversity of flower visitors varied between all eight sites. Site one had low richness an d diversity. Perhaps this finding is due to the fact that site one was in a high risk area, pasture, and Ithomiines tend to avoid these areas. Species richness and diversity was also much lower in females comparative to males but when comparing

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7 richness and diversity among females, results were the same. These may be account for by the small sample. so. This suggests that females prefer to remain in safe forest areas, leks, from males while mating. This therefore forces males to leave the safety of the forest and replenish PA sources lost to pheromones, mating, and defense. In future studies, it may be useful to observe female Ithomiine behavior within the c onfines of leks containing A. reedii . It would also prove effective to find more than one lek of Ithomiinae in order for comparison in both visitation patterns of males and females as well as general abundance between different populations. Unknown still , and noteworthy is whether only particular species of Ithomiinae females visit A. reedii . ACKNOWLEDGEMENTS I would like to first thank Alan Masters for his continual support and encouragement even when my first project failed. He has inspired me to c ontinue with science despite the many frustrations and initial failures that come hand and hand with the field. A special thanks to the San Luis EcoLodge. Their forest provided countless hours of enjoyment even when data collection seemed endless. I wou ld also like to thank both Oliver Hymen and Matt Gasner, our teaching assistants, for their help finding sources, formatting, and general support during the final report. Special thanks to Karen Masters for her loving support through everything. A final thanks to William Haber and Willow Zuchowski for their extensive knowledge regarding butterflies and plants. LITERATURE CITED Brown, K.S. Jr. 1984. Adult obtained pyrrolizidine alkaloids defend ithomiine butterflies against a spider predator. Natur e 309: 707 709. Brown, K.S. Jr., Jose R. Trigo, Ronald B. Francini, Ana Beatriz Barros de Morais, and Paulo C. Motta. 1991. Aposematic Insects on Toxic Host Plants: Coevolution, Colonization, and Chemical Emancipation. In. Price, P., W. Benson, G. Fern andes, and T. Lewinsohn. Plant Animal Interactions, Evolutionary Ecology in Tropical and Temperate Regions. DeVries, P. J. 1987. The Butterflies of Costa Rica, and Their Natural History. Princeton University Press, Chichester West Sussex. Drummond, B. A. III. 1976. Comparative Ecology and Mimetic Relationships of Ithomiine Butterflies in Eastern Ecuador. PhD. dissertation. University of Florida. Haber, W. A. 1978. Evolutionary Ecology of Tropical Mimetic Butterflies (Lepidoptera: Ithomiinae) . PhD. dissertation. University of Minnesota. Hayes, M. and R. Laval. 1989. The mammals of Monteverde. 2 nd edition. San Jose, Costa Rica.

9 Figure 1 . Three different measurements of Ithomiinae diversity across eight sites in San Luis, Costa Rica for both male and female species of Ithomiinae (a) abundance (b) species richness (c) Shannon Wiener diversity indices. Sites were located in pasture (1), road (2,8), secondary forest (3,4), and primary forest (5 7). Site four repre sents the center of an Ithomiinae lek. Counts are for Ithomiines captured at A. reedii bates placed in each locale 1.5 meters off the ground. c b a

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10 Table 1 . Sorenson indices overlap matrix between eight sites in San Luis, Costa Rica. The more species two sites have in common the closer the index is to one. Sites were located in pasture (1), road (2, 8), secondary forest (3,4), and primary forest (5 7). Site four also represents the center of an Ithomiinae lek. Male and female Ithomiinae species were ob served at each site and recorded when they visited A. reedii , which were placed in bundles at each site approximately 1.5 meters off the ground. Sites 1 2 3 4 5 6 7 8 Sites 1 X 0.207 0.207 0.072 0.113 0.136 0.133 0.068 2 X 0.471 0.377 0.500 0.62 7 0.528 0.198 3 X 0.574 0.696 0.675 0.609 0.315 4 X 0.600 0.769 0.649 0.326 5 X 0.725 0.701 0.326 6 X 0.559 0.222 7 X 0.196 8 X

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11 APPENDIX Figure 2. Distances to sites measured from the center of an Ithomiinae lek found in San Luis, Costa Rica. Site four represents the center of the lek. The lek boundaries are represented by the black oval. Sites three and five are on the edge of the lek. Sites were located in pasture (1), road (2, 8), second ary forest (3,4), and primary forest (5 7). Table 2. Name of Ithomiinae species, number of male to females of each species found, and total number of male to female found within eight sties in San Luis, Costa Rica. . Sites were located in pasture ( 1), road (2, 8), secondary forest (3,4), and primary forest (5 7). Site four also represents the center of an Ithomiinae lek. Male and female Ithomiinae species were observed at each site and recorded when they visited A. reedii , which had been strategic ally placed in bundles at each site approximately one and a half meters from the ground in light gaps. Fractions are set up as male over female for each site and species